ROLE OF PDI FAMILY PROTEINS FOR FOLDING OF IMMUNOGLOBULINS

PDI 家族蛋白在免疫球蛋白折叠中的作用

• 批准号: 09670249
• 负责人: SAGA Shinsuke
• 金额: $ 0.64万
• 依托单位: AICHI MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670249/
• 关键词: PDI; ERp61; ERp72; protein folding; immunoglobulins; molecular chaperones; deletion mutants; S-S結合

Folding and assembly of nascent polypeptides involves two kinds of proteins, molecular chaperones and the enzymes that catalyze the formation of covalent bonds. The representative of the latter is protein disulfide isomerase (PDI) that is a resident protein in the endoplasmic reticulum (ER) with thioredoxin-like domains containing active sites (CGHC) responsible for its redox activity. Similar structures are also present in ERp61 and ERp72, other ER resident proteins. These PDI family proteins are implicated in the disulfide bond formation of nascent polypeptides in the ER lumen.In this study, the effect of PDI family proteins on reconformation of reduced and denatured immunoglobulins was examined, It was failed to detect the difference of their activity for in vitro reconformation of IgG.Unfortunately, it was very difficult to establish in vitro reconformation system for IgM and IgA due to the low solubility of denatured immunoglobulins. Western blotting of immuno-precipitates with an … More ti- alpha chain or anti- mu chain antibody from cell lysate crosslinked in situ with dithiosuccinimidyl propionate (DSP) elucidated the association of ERp61 with IgM and of ERp72 with IgA and IgM, but no association of PDI with both immunoglobulins was detected. These results indicate that different enzymes may catalyze the disulfide bond formation in different class of immunoglobulins.In order to analyze the chaperone activity of ERp72, delution mutants of ERp72 were expressed as histidine taged proteins in E coli and purified, Analysis of their activity of binding to denatured insulin peptides and immunoglobulins is on going.The expression of PDI family proteins in mouse F9 teratocarcinoma cells during differentiation induced with retinoic acid and dibutyryl cAMP.The differentiation clearly induced all these enzymes. In immunoprecipitation experiments combined with in situ chemical crosslinking, type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Less

新生多肽的折叠和组装涉及两种蛋白质，分子伴侣和催化共价键形成的酶。后者的代表是蛋白质二硫键异构酶（PDI），其是内质网（ER）中的驻留蛋白，具有含活性位点的硫氧还蛋白样结构域（CGHC），负责其氧化还原活性。类似的结构也存在于其他ER驻留蛋白ERp61和ERp72中。这些PDI家族蛋白参与ER腔中新生多肽的二硫键形成。在本研究中，检测了PDI家族蛋白对还原和变性免疫球蛋白的重构的作用，未能检测到它们对IgG体外重构的活性差异。不幸的是，由于变性免疫球蛋白溶解度低，很难建立IgM和伊加的体外重组体系。免疫沉淀物的Western印迹， ...更多信息 来自与二硫代琥珀酰亚胺基丙酸酯（DSP）原位交联的细胞裂解物的α链或μ链抗体阐明了ERp61与IgM的关联以及ERp72与伊加和IgM的关联，但未检测到PDI与两种免疫球蛋白的关联。为了分析ERp72的分子伴侣活性，我们在大肠杆菌中表达了ERp72的缺失突变体，并将其纯化为组氨酸标签蛋白，对它们与变性胰岛素肽和免疫球蛋白的结合活性的分析正在进行中。用维甲酸和双丁酰cAMP诱导分化。分化明显诱导所有这些酶。在结合原位化学交联的免疫沉淀实验中，IV型胶原蛋白与PDI显着共沉淀，而层粘连蛋白同样与三种蛋白质共沉淀。此外，210 kDa的蛋白质特征性地与ERp72共沉淀。少

项目成果

Kojima, T.et al.: "The retention of abnormal type I procollagen and correlated expression of HSP 47 in fibroblasts from a patient with lethal osteogenesis imperfecta." Journal of Pathology. 184. 212-218 (1998)

Kojima, T. 等人：“致命性成骨不全患者的成纤维细胞中异常 I 型前胶原的保留和 HSP 47 的相关表达。”

Kato, K.et al.: "Phosphorylation of alpha B-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation." Journal of Biological Chemistry. 273. 28346-28354 (1998)

Kato, K. 等人：“有丝分裂细胞中 α B-晶状体蛋白的磷酸化以及负责磷酸化的酶活性的鉴定。”

Kozaki, K.: "Isolation, purification and characterization of a collagen-associated serpin, Caspin, produced by murine colon adenocarcinoma cells." Journal of Biological Chemistry. 273. 15125-15130 (1998)

Kozaki, K.：“小鼠结肠腺癌细胞产生的胶原相关丝氨酸蛋白酶抑制剂 Caspin 的分离、纯化和表征。”

Kojima,T.: "The retention of abnormal type I procollagen and correlated expression of HSP 47 in fibroblasts from a patient with lethal osteogenesis imperfecta." Journal of Pathology. 184. 212-218 (1998)

Kojima,T.：“致命性成骨不全患者的成纤维细胞中异常 I 型前胶原的保留和 HSP 47 的相关表达。”

Miyaishi, O.et al.: "Elevated expression of PDI family proteins during differentiation of mouse F9 teratocarcinoma cells" Journal of Cellular Biochemistry. 68. 436-445 (1998)

Miyaishi, O.等人：“小鼠 F9 畸胎癌细胞分化过程中 PDI 家族蛋白的表达升高”《细胞生物化学杂志》。